Detection of polymorphisms of human DNA by gel electrophoresis as SSCPs

Abstract

We developed mobility shift analysis of single-stranded DNAs on neutral polyacrylamide gel electrophoresis to detect DNA polymorphisms. This method follows digestion of genomic DNA with restriction endonucleases, denaturation in alkaline solution, and electrophoresis on a neutral polyacrylamide gel. After transfer to a nylon membrane, the mobility shift due to a nucleotide substitution of a single-stranded DNA fragment could be detected by hybridization with a nick-translated DNA fragment or more clearly with RNA copies synthesized on each strand of the DNA fragment as probes. As the mobility shift caused by nucleotide substitutions might be due to a conformational change of single-stranded DNAs, we designate the features of single-stranded DNAs as single-strand conformation polymorphisms (SSCPs). Like restriction fragment length polymorphisms (RFLPs), SSCPs were found to be allelic variants of true Mendelian traits, and therefore they should be useful genetic markers. Moreover, SSCP analysis has the advantage over RFLP analysis that it can detect DNA polymorphisms and point mutations at a variety of positions in DNA fragments. Since DNA polymorphisms have been estimated to occur every few hundred nucleotides in the human genome, SSCPs may provide many genetic markers.

Full text

Proc.
Natd.
Acad.
Sci.
USA
Vol.
86,
pp.
2766-2770,
April
1989
Genetics
Detection
of
polymorphisms
of
human
DNA
by
gel
electrophoresis
as
single-strand
conformation
polymorphisms
(mobility
shift
of
separated
strands/point
mutation/riction
franment
length
polymorphism)
MASATO
ORITA,
HIROYUKI
IWAHANA*,
HIROSHI
KANAZAWAt,
KENSHI
HAYASHI,
AND
TAKAO
SEKIYA*
Oncogene
Division,
National
Cancer
Center
Research
Institute,
Tsukiji
5-1-1,
Chuo-ku,
Tokyo
104,
Japan
Communicated
by
Takashi
Sugimura,
December
29,
1988
ABSTRACT
We
developed
mobility
shift
analysis
of
sin-
gle-stranded
DNAs
on
neutral
polyacrylamide
gel
electropho-
resis
to
detect
DNA
polymorphisms.
This
method
follows
digestion
of
genomic
DNA
with
restriction
endonucleases,
denaturation
in
alkaline
solution,
and
electrophoresis
on
a
neutral
polyacrylamide
gel.
After
transfer
to
a
nylon
mem-
brane,
the
mobility
shift
due
to
a
nucleotide
substitution
of
a
single-stranded
DNA
fragment
could
be
detected
by
hybrid-
ization
with
a
nick-translated
DNA
fragment
or
more
clearly
with
RNA
copies
synthesized
on
each
strand
of
the
DNA
fragment
as
probes.
As
the
mobility
shift
caused
by
nucleotide
substitutions
might
be
due
to
a
conformational
change
of
single-stranded
DNAs,
we
designate
the
features
of
single-
stranded
DNAs
as
single-strand
conformation
polymorphisms
(SSCPs).
Like
restriction
fragment
length
polymorphisms
(RFLPs),
SSCPs
were
found
to
be
allelic
variants
of
true
Mendelian
traits,
and
therefore
they
should
be
useful
genetic
markers.
Moreover,
SSCP
analysis
has
the
advantage
over
RFLP
analysis
that
it
can
detect
DNA
polymorphisms
and
point
mutations
at
a
variety
of
positions
in
DNA
fragments.
Since
DNA
polymorphisms
have
been
estimated
to
occur
every
few
hundred
nucleotides
in
the
human
genome,
SSCPs
may
provide
many
genetic
markers.
The
nucleotide
sequences
of
DNAs
in
humans
are
not
identical
in
different
individuals.
Nucleotide
substitutions
have
been
estimated
to
occur
every
few
hundred
base
pairs
in
the
human
genome
(1).
Nucleotide
sequence
polymor-
phism
has
been
detected
as
restriction
fragment
length
polymorphism
(RFLP).
RFLP
analysis
of
family
members
has
been
used
to
construct
a
genetic
linkage
map
of
the
human
genome
(2,
3),
and
this
analysis
has
also
revealed
the
chromosomal
locations
of
genetic
elements
involved
in
he-
reditary
diseases
such as
Huntington
disease
(4),
adult
polycystic
kidney
disease
(5),
cystic
fibrosis
(6-8),
Alzhei-
mer
disease
(9,
10),
and
Duchenne
muscular
dystrophy
(11,
12).
Thus
prenatal
diagnosis
of
diseases
such
as
cystic
fibrosis
is
possible
with
RFLP
probes.
Recently,
RFLP
analysis
has
indicated
specific
loss
of
heterozygosity
at
particular
loci
on
chromosomes
in
cancerous
portions
of
tissues
in
several
human
cancers,
including
retinoblastoma,
Wilms
tumor,
small
cell
carcinoma
of
the
lung,
renal
cell
carcinoma,
bladder
carcinoma,
breast
carcinoma,
meningioma,
acoustic
neuroma
(see
ref.
13
for
a
review),
colorectal
carcinoma
(14,
15),
and
multiple
endocrine
neoplasia
type
1-
or
type
2-
associated
carcinomas
(16,
17).
This
loss
of
heterozygosity
suggests
the
involvement
of
recessive
mutation
of
particular
genes
in
development
of
these
cancers.
Although
RFLPs
are
very
useful
for
distinguishing
two
alleles
at
chromosomal
loci,
they
can
be
detected
only
when
DNA
polymorphisms
are
present
in
the
recognition
se-
quences
for
the
corresponding
restriction
endonucleases
or
when
deletion
or
insertion
of
a
short
sequence
is
present
in
the
region
detected
by
a
particular
probe.
To
identify
DNA
polymorphisms
more
efficiently,
Noll
and
Collins
used
a
simplified
method
of
denaturing
gradient
gel
electrophoresis
(18)
that
had
been
developed
by
Myers
et
al.
(19).
As
analysis
of
mobility
shift
[probably
due
to
a
conformational
change
of
single-stranded
DNAs
on
polyacrylamide
gel
electrophoresis
(20)]
has
been
used
to
detect
point
mutations
(21),
in
this
work
we
examined
whether
the
mobility
shift
of
single-stranded
DNA
caused
by
a
single
nucleotide
substitution
could
be
used
to
detect
nucleotide
sequence
polymorphisms.
The
results
indicated
that
mobility
shift
analysis
is
an
efficient
method
for
detecting
DNA
polymorphisms
and
for
distinguishing
the
two
alleles
at
chromosomal
loci.
MATERIALS
AND
METHODS
Cell
Lines.
The
human
bladder
carcinoma
cell
line
T24
was
obtained
from
the
American
Type
Culture
Collection.
The
human
malignant
melanoma
cell
line
SK2
was
established
from
a
tissue
that
had
been
maintained
in
nude
mice
(22).
DNA
Isolation.
High
molecular
weight
DNA
was
prepared
from
human
leukocytes
or
cultured
human
tumor
cell
lines
by
the
method
of
Blin
and
Stafford
(23).
Plasmids.
Plasmid
pNCO106
was
prepared
by
inserting
a
2.9-kilobase
pair
(kb)
Sac
I
fragment
of
the
HRASI
gene
from
SK2
cells
into
pUC19
(24).
Plasmid
pT22
was
constructed
by
inserting
a
6.6-kb
BamHI
fragment
of
the
HRASI
gene
from
T24
cells
into
pBR322
(a
gift
from
M.
Wiglar,
Cold
Spring
Harbor
Laboratory).
Subcloning
and
Sequencing
of
DNA
Fragments.
From
pNCO1O6
and
pT22,
a
371-base-pair
(bp)
Pst
I
fragment
carrying
exon
1
and
a
298-bp
Pst
I
fragment
containing
exon
2
of
the
HRASI
gene
were
isolated
and
subcloned
into
the
pGEM-2
vector
(Promega
Biotec).
The
nucleotide
sequences
of
the
subcloned
fragments
were
determined
by
the
dideoxy-
nucleotide
method
(25),
using
Sequenase
(United
States
Biochemical)
and
the
SP6
or
T7
promoter
primer
(Promega
Biotec).
Analysis
of
Single-Strand
Conformation
Polymorphisms
(SSCPs).
High
molecular
weight
DNA
(20
jig)
was
digested
completely
with
restriction
endonucleases
under
the
condi-
tions
recommended
by
the
suppliers.
The
reaction
mixture
was
extracted
once
with
phenol/chloroform
(1:1,
vol/vol)
and
once
with
chloroform.
After
addition
of
0.1
vol
of
3
M
sodium
acetate,
DNA
fragments
were
precipitated
from
the
Abbreviations:
RFLP,
restriction
fragment
length
polymorphism;
SSCP,
single-strand
conformation
polymorphism.
*Present
address:
Department
of
Internal
Medicine,
School
of
Medicine,
The
University
of
Tokushima,
3-18-15
Kuramoto-cho,
Tokushima
770,
Japan.
tPresent
address:
Department
of
Applied
Biology,
Faculty
of
Tech-
nology,
Okayama
University,
3-1-1
Tsushimanaka,
Okayama
700,
Japan.
*To
whom
reprint
requests
should
be
addressed.
2766
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertisement"
in
accordance
with
18
U.S.C.
§1734
solely
to
indicate
this
fact.

Proc.
Natl.
Acad.
Sci.
USA
86
(1989)
2767
aqueous
phase
by
addition
of
2.5
vol
of
ethanol.
Strands
were
separated
out
by
the
method
of
Maxam
and
Gilbert
(20)
with
a
slight
modification.
DNA
precipitates
were
dissolved
in
20
Al
of
denaturing
solution
(0.3
M
NaOH/1
mM
EDTA)
and
then
mixed
with
3
Al
of
50%
(vol/vol)
glycerol/0.25%
xylene
cyanol/0.25%
bromophenol
blue.
The
mixture
was
applied
to
a
neutral
5%
polyacrylamide
gel
(20
x
40
x
0.2
cm)
with
or
without
10%
glycerol
in
a
well
of
10
mm
width
and
subjected
to
electrophoresis
in
90
mM
Tris-borate,
pH
8.3/4
mM
EDTA
at
180
V
for
12-36
hr
at
17°C.
DNA
fragments
in
the
gel
were
then
transferred
to
a
nylon
membrane
(Hybond-N,
Amer-
sham)
by
electrophoretic
blotting
in
0.025
M
sodium
phos-
phate,
pH
6.5,
at
1
A
for
2
hr
at
4°C
by
the
procedure
recommended
by
the
membrane
supplier.
The
membrane
was
then
dried
and baked
at
80°C
for
2
hr.
Hybridization
with
32P-labeled
DNA
probes
was
performed
in
50%
(vol/vol)
formamide/6x
SSC
(lx
SSC
is
0.15
M
sodium
chloride/
0.015
M
sodium
citrate,
pH
7.0)/10
mM
EDTA/5x
Den-
hardt's
solution
(lx
Denhardt's
solution
is
0.02%
bovine
serum
albumin/0.02%
Ficoll/0.02%
polyvinylpyrrolidone)/
0.5%
NaDodSO4
containing
denatured
salmon
sperm
DNA
at
100
jg/ml
and
10%
dextran
sulfate
at
42°C
for
16
hr.
The
blots
were
washed
twice
in
2x
SSC/0.1%
NaDodSO4
for
30
min
at
65°C
and
then
once
in
0.1
x
SSC
at
65°C
for
10
min.
Autoradiography
was
carried
out
at
-80°C
for
2-7
days
by
exposing
the
membranes
to
x-ray
film
(XAR-5,
Kodak)
with
an
intensifying
screen
(Cronex
Lightning
Plus,
DuPont).
Analysis
of
RFLP.
RFLP
analysis
was
performed
as
de-
scribed
(26).
High
molecular
weight
DNA
(5
jig)
was
digested
with
an
appropriate
restriction
endonuclease
and
the
digest
was
fractionated
by
electrophoresis
in
a
0.7%
agarose
gel.
DNA
Probes
for
Hybridization.
Cloned
Pst
I
fragments
371
and
298
bp
long
carrying
exon
1
and
2
of
the
normal
human
HRASJ
gene
(27),
respectively,
were
used
as
specific
probes
for
the
corresponding
exons.
The
2.8-kb
HindIII
fragment
isolated
from
phage
9D11
(28),
provided
by
the
Japanese
Cancer
Research
Resources
Bank,
was
used
as
a
specific
probe
for
the
D13S2
locus
on
human
chromosome
13
(29).
Probes
were
labeled
to
a
specific
activity
of
2-10
x
108
cpm/,ug
by
nick-translation
(30)
with
[a-32P]dCTP
(3000
Ci/mmol;
1
Ci
=
37
GBq)
as
a
radioactive
substrate.
RNA
Probes
for
Hybridization.
Single-stranded
RNA
probes
were
prepared
by
the
method
of
Melton
et
al.
(31)
with
plasmid
constructs
carrying
the
fragments
used
as
DNA
probes
in
the
pGEM-2
vector
as
templates.
RNA
synthesis
on
each
strand
of
the
templates
was
carried
out
with
T7
RNA
polymerase
(TOYOBO,
Tokyo)
or
SP6
RNA
polymerase
(Amersham)
in
the
presence
of
[a-32P]UTP
as
a
radioactive
substrate.
Concentration
of
UTP
was
adjusted
to
500
,M
by
adding
the
nonradioactive
nucleotide
(final
specific
activity,
40
Ci/mmol)
to
ensure
synthesis
of
full-length
RNA
copies.
The
hybridization
conditions
and
washing
procedures
were
the
same
as
those
for
DNA
probes.
RESULTS
Mobility
Shift
by
Single
Base
Substitution.
To
determine
whether
a
single
base
substitution
altered
the
mobility
of
single-stranded
DNAs
on
neutral
polyacrylamide
gel
electro-
phoresis,
we
separated
Pst
I
fragments
carrying
exon
1
or 2
of
the
human
HRASJ
gene,
whose
nucleotide
sequences
are
known.
In
the
human
melanoma
cell
line
SK2,
one
of
the
two
alleles
of
the
HRASJ
gene
is
known
to
be
activated
by
point
mutation
at
codon
61
in
exon
2
(32)
and
also
amplified
about
10-fold
(33).
The
human
bladder
carcinoma
cell
line
T24
has
been
reported
to
contain
only
one
allele
of
the
HRASI
gene,
which
carries
a
mutated
codon
12
in
exon
1
(34,
35).
From
plasmid
constructs
pNCO106
and
pT22,
containing
the
trans-
forming
allele
of
the
HRASI
gene
of
SK2
and
T24
cells,
respectively,
a
371-bp
Pst
I
fragment
carrying
exon
1
of
the
gene
was
isolated
and
subcloned
in
the
pGEM-2
vector.
Similarly,
a
298-bp
Pst
I
fragment
carrying
exon
2
of
the
HRASI
gene
was
isolated
from
the
same
plasmid
constructs
and
subcloned.
By
determination
of
the
total
nucleotide
sequences
of
the
subcloned
fragments,
we
confirmed
the
single
nucleotide
substitution
at
codon
12
in
the
371
nucleo-
tides
of
the
Pst
I
fragment
between
the
SK2
gene
and
the
T24
gene
(GGC
in
the
SK2
gene
and
GTC
in
the
T24
gene).
The
nucleotide
sequences
of
the
298-bp
Pst
I
fragments
carrying
exon
2
of
the
SK2
and
T24
genes
were
also
confirmed
to
differ
from
each
other
by
only
one
nucleotide
in
codon
61
(CTG
in
the
SK2
gene
and
CAG
in
the
T24
gene).
After
denaturation
in
alkaline
solution,
these
cloned
Pst
I
fragments
were
subjected
to
electrophoresis
in
neutral
5%
polyacrylamide
gel.
The
separated
strands
were
then
transferred
to
a
nylon
membrane
by
electrophoretic
blotting
and
hybridized
with
32P-labeled
DNA
probes.
As
shown
in
Fig.
LA,
the
pair
of
separated
strands
of
the
Pst
I
fragment
carrying
exon
1
of
the
T24
gene
(lane
2)
moved
slightly
faster
than
those
of
the
SK2
gene
(lane
1).
In
the
case
of
the
Pst
I
fragment
carrying
exon
2,
the
mobilities
of
the
separated
strands
of
the
SK2
gene
(Fig.
LA,
lane
3)
were
significantly
different
from
those
of
the
T24
gene
(lane
4).
Three
bands
were
observed
in
the
sample
from
the
SK2
gene.
Hybridization
with
single-stranded
RNA
probes
showed
that
the
bands
with
the
fastest
and
the
slowest
mobilities
were
from
the
same
strand
of
the
fragment,
while
the
middle
band
corresponded
to
the
complementary
strand
(data
not
shown).
Usually
the
slowest-moving
band
was
the
major
one
from
the
particular
strand
and
the
ratio
of
the
slowest
and
the
fastest
bands
varied
depending
on
the
conditions
of
electrophoresis,
especially
the
temperature
of
the
running
gels.
These
results
suggested
that
a
particular
single-stranded
DNA
could
take
at
least
two
different
mo-
lecular
shapes,
depending
on
the
conditions
of
electropho-
resis.
In the
system
containing
homogeneous
cloned
DNA
frag-
ments,
we
could
demonstrate
mobility
shift
of
single-
stranded
DNAs
due
to
a
single
base
substitution.
To
deter-
mine
whether
the
same
mobility
shift
could
be
observed
in
the
presence
of
DNA
fragments
other
than
a
target
fragment,
we
digested
genomic
DNAs
from
the
two
tumor
cell
lines
SK2
and
T24
with
Pst
I
and
subjected
the
total
digests
to
electrophoresis
in
neutral
polyacrylamide
gel
after
denatur-
ation.
As
shown
in
Fig.
1B,
the
patterns
of
the
separated
strands
of
the
fragments
carrying
exon
1
or
2
of
the
HRASI
gene
from
the
genomic
DNAs
were
essentially
the
same
as
those
of
the
cloned
fragments.
This
result
indicated
that
the
mobility
shift
due
to
a
single
base
substitution
of
a
single-
stranded
DNA
fragment
in
total
digests
of
genomic
DNA
A
1
2
3
4
B
1
2
3
4
FIG.
1.
Mobility
shift
of
single-stranded
DNA
fragments
due
to
a
single
base
substitution.
(A)
Plasmid
clones
(2
pg)
of
fragments
carrying
exon
1(371
bp)
and
exon
2
(298
bp)
of
the
HRASI
gene
from
malignant
melanoma
SK2
cells
(lanes
1
and
3,
respectively)
and
from
bladder
carcinoma
T24
cells
(lanes
2
and
4,
respectively)
were
digested
with
Pst
I.
(B)
Total
genomic
DNAs
(20
,ug)
from
SK2
cells
(lanes
1
and
3)
and
from
T24
cells
(lanes
2
and
4)
were
digested
with
Pst
I.
After
denaturation,
the
fragments
produced
were
subjected
to
electrophoresis
in
neutral
polyacrylamide
gel
without
glycerol.
Single-stranded
DNAs
were
transferred
to
a
nylon
membrane
and
hybridized
with
the
32P-labeled
DNA
probe
for
exon
1
of
the
HRASI
gene
(lanes
1
and
2
in
A
and
B)
and
the
probe
for
exon
2
of
the
gene
(lanes
3
and
4
in
A
and
B).
Genetics:
Orita
et
al.
..A.
W
:....:..-A
W-d
%M.*
*..*
"
1
IIWW40
a"

Proc.
NatL.
Acad.
Sci.
USA
86
(1989)
could
be
detected
and
was
not
influenced
by
the
presence
of
a
large
amount
of
unrelated
DNA
fragments.
SSCP
Analysis
of
Human
DNA
at
the
D13S2
Locus.
The
above
results
encouraged
us
to
apply
the
mobility
shift
of
single-stranded
DNA
due
to
a
single
base
substitution
to
detection
of
nucleotide
sequence
polymorphisms
of
a
partic-
ular
fragment
and,
as
can
be
done
with
RFLPs,
to
distin-
guishing
two
alleles
at
chromosomal
loci.
As
the
mobility
shift
might
be
due
to
a
conformational
change
of
the
single-
stranded
DNAs,
we
designated
the
polymorphisms
detected
by
the
method
as
SSCPs.
Leukocyte
DNA
samples
from
19
individuals
(10
unrelated
and
9
in
two
families)
were
digested
with
Hae
III,
and
SSCPs
of
the
fragments
obtained
from
a
region
of
about
3
kb
at
the
D13S2
locus
on
chromosome
13
were
analyzed.
When
the
digests
were
subjected
to
electrophoresis
without
denatur-
ation
and
hybridized
with
the
32P-labeled
2.8-kb
HindIII
fragment
as
a
specific
probe
for
the
D13S2
locus,
five
distinct
double-stranded
DNA
fragments
(F1
to
F5
in
order
of
size)
without
any
RFLP
were
observed
in
all
DNA
samples.
The
results
on
DNA
samples
1
and
2
are
shown
in
Fig.
2A
as
examples.
In
contrast
with
the
double-stranded
fragments,
separated
strands
of
the
same
DNA
fragments
showed
SSCPs
with
considerable
frequency.
Representative
results
are
shown
in
Fig.
2
B-D.
When
nick-translated
DNA
was
used
as
a
probe,
SSCPs
were
apparently
observed
in
at
least
one
of
the
four
fragments
(F2
to
F5)
in
all
four
DNA
samples
(Fig.
2B).
The
mobility
shift
of
one
of
the
strands
of
fragment
F4
in
sample
1
was
especially
marked.
However,
the
mobility
shifts
of
singe
strands
in
other
fragments
were
small
and
therefore
the
difference
of
the
shifts
was
not
clear
when
both
strands
of
the
fragments
were
hybridized
with
the
nick-
translated
probe.
To
overcome
this
disadvantage,
RNA
copies
(RNA
1
and
2
in
Fig.
2
C
and
D)
of
each
strand
of
the
D13S2
DNA
fragment
were
prepared
separately
and
used
as
probes
for
hybridization.
As
shown
in
Fig.
2
C
and
D,
with
either
the
RNA
1
or
RNA
2
probe
SSCPs
were
clearly
detected
in
all
fragments
except
fragment
Fl.
In
Fig.
2E,
the
alleles
distinguished
by
SSCPs
are
summarized.
SSCPs
found
in
fragment
F2
by
using
the
RNA
1
probe
could
distinguish
alleles
with
three
different
mobilities,
designated
as
"slow"
(s),
"fast"
(f),
and
"very
fast"
(vf).
In
addition
to
these
three
A
bp
1
2
1200--
-
alleles,
the
SSCP
analysis
of
the
other
DNA
sample
shown
in
Fig.
3A
revealed
the
presence
of
an
allele
with
"very
slow"
(vs)
mobility
in
the
fragment.
The
SSCPs
of
the
other
fragments,
F3, F4,
and
F5,
could
also
distinguish
at
least
two
alleles
with
"slow"
(s)
or
"fast"
(f)
mobility.
Analysis
of
19
DNA
samples
revealed
that
mobility
shifts
found
in
F4
and
F5
were
coincidental.
Mendelian
Inheritance
of
SSCPs.
To
confirm
that
the
observed
SSCPs
of
the
Hae
III
fragments
of
the
region
at
the
D13S2
locus
were
due
to
allelic
variants
of
true
Mendelian
traits,
we
analyzed
the
DNAs
of
nine
individuals
in
two
related
families.
In
Fig.
3A,
SSCPs
of
fragments
F2,
F3,
and
F4
and
the
alleles
identified
are
indicated.
In
each
family,
the
genotypes
of
the
progenies
were
consistent
with
the
parental
genotypes.
Relationship
Between
SSCPs
and
RFLPs.
The
same
19
DNA
samples
analyzed
for
SSCPs
were
also
subjected
to
RFLP
analysis.
The
DNAs
were
digested
with
Msp
I
or
Taq
I
and
RFLPs
were
detected
by
hybridization
with
the
32P-labeled
DNA
probe
for
the
D13S2
locus.
Of
the
19
DNA
samples
digested
with
Msp
I,
five
samples
(sample
2
in
Fig.
2,
data
not
shown,
samples
2,
3,
5,
and
8
in
Fig.
3B)
showed
RFLP.
By
Taq
I
digestion,
RFLP
was
observed
in
only
one
of
the
DNA
samples
(sample
2
in
Fig.
2,
data
not
shown).
Therefore,
RFLP
analysis
revealed
heterozygosity
at
the
D13S2
locus
in
only
5
of
19
individuals,
while
with
SSCP
analysis
heterozy-
gosity
at
the
locus
was
found
in
at
least
one
of
the
four
Hae
III
fragments
in
18
of
the
19
DNA
samples.
This
fact
demonstrates
that
SSCP
analysis
is
a
superior
tool
for
detection
of
genetic
polymorphisms.
Factors
Affecting
SSCP
Analysis.
The
mobility
shift
of
single-stranded
DNAs
with
DNA
polymorphisms
observed
on
neutral
polyacrylamide
gel
electrophoresis
is
most
likely
due
to
conformational
variations
of
the
molecules.
The
conformation
of
single-stranded
nucleic
acid
is
expected
to
be
affected
by
environmental
factors
such
as
the
temperature
of
the
gel
during
electrophoresis,
the
concentration
of
electro-
phoresis
buffer,
and
the
presence
of
denaturing
agents
in
gels.
The
mobility
shift
of
the
Pst
I
fragments
carrying
exon
1
of
the
HRASI
gene
shown
in
Fig.
1A
(lanes
1
and
2)
was
clearly
observed
on
electrophoresis
at
17°C
but
not
prominently
at
23°C
(data
not
shown).
The
pattern
of
the
separated
strands
B
C
D
E
1
2
34
1
2
34
1
2 3
4
Fragment
-
Probe
1
2
3
4
F2/RNA1m
=
mi
s/f
s/vf
s/f
f/vf
F2
mmm
m_
-
-
F3
-
.
W
F
4
-O
-
m
-
F3/RNAl
__
_11W
4okwk_
*.
,
f/f
s/f
s/s
s/f
F4/RNA2
s/f
s/s
s/s
s/s
310-
F5
Probe:
dsDNA
RNA1
RNA2
F5/RNA1
s/f
s/s
s/s
s/s
FIG.
2.
SSCP
analysis
of
human
DNAs
at
the
D13S2
locus.
DNA
samples
1-4
were
prepared
from
leukocytes
of
four
unrelated
individuals
and
digested
with
Hae
III.
The
resultant
fragments
were
subjected
to
electrophoresis
in
neutral
polyacrylamide
gel
containing
1o
glycerol
before
(A)
and
after
(B,
C,
and
D)
denaturation.
DNAs
in
the
gel
were
transferred
to
a
nylon
membrane
and
then
hybridized
with
the
32P-labeled
double-stranded
DNA
(dsDNA)
probe
for
the
D13S2
locus
(A
and
B)
and
with
the
32P-labeled
single-stranded
RNA
probes
for
the
D13S2
locus
(RNA1
in
C
and
RNA2
in
D).
The
five
fragments
produced
from
the
D13S2
region
by
Hae
III
digestion
were
designated
as
F1
to
F5
in
order
of
size.
Alleles
identifed
by
SSCPs
are
indicated
in
E
with
higher
magnifications
of
informative
fragments
observed
in
C
or
D.
650-
-
440-
430-
2768
Genetics:
Orita
et
al.

Proc.
Natl.
Acad.
Sci.
USA
86
(1989)
2769
A
Fragment
Probe
F2/RNA1
1
2
3
4
5
6
7
:A
.Wm-
I
s/vf
f/tV
vt/vt
s/f
vs/vt
f/f
f/V
F3/RNA1
_
-M
_ -
s/f
s/f
f/f
s/s
s/f
s/f
s/
F4/RNA2
s/f
s/s
s/s
s/f
s/s
s/s
s/
B
kb
1
2
3
4
5
6
7
8
9
15-
W-
w
w
w
w
10.5-
_w
"
w
FIG.
3.
SSCP
and
RFLP
analyses
of
family
Leukocyte
DNAs
(20
lug)
from
the
family
members
i
top
(o,
females;
i,
males)
were
subjected
to
SSCP
an'
D13S2
probe
as
described
in
the
legend
for
Fig.
2.
)
shifts
found
in
fragments
F4
and
F5
were
the
same,
t
fragment
F5
are
not
shown.
(B)
The
leukocyte
DNAs
with
Msp
I
were
subjected
to
RFLP
analysis
usi
dsDNA
as
a
probe
for
the
D13S2
locus.
of
the
fragments
carrying
exon
2
of
the
gene
obs
and
shown
in
Fig.
1A
(lanes
3
and
4)
was
also
al
Thus,
the
higher
temperature
might
destroy
sor
conformations.
The
concentration
of
the
runnih
affected
the
mobility
shift.
When
electrophores
fragments
analyzed
in
Fig.
1A
was
performed
lower
concentration
(45
mM
Tris-borate,
p]
EDTA)
at
17TC,
the
mobility
shifts
observed
w
those
at
the
higher
temperature
(230C).
Pres
glycerol
in
gels
also
affected
the
mobility
shift.
effect
of
glycerol
was
rather
complicated
and
n
due
to
DNA
polymorphisms
were
often
enha
reagent.
For
example,
the
mobility
shifts
obse]
were
enhanced
when
electrophoresis
was
per]
containing
10%
glycerol.
On
the
other
hand,
the
shown
in
Fig.
1
was
reduced
by
the
presence
of
in
the
gel.
DISCUSSION
By
neutral
polyacrylamide
gel
electrophores
separate
two
single-stranded
DNA
fragments
nucleotide
sequences
differed
at
only
one
I
mobility
shift
due
to
a
single
base
substitut
observed
not
only
in
cloned
fragments
but
also
of
total
genomic
DNA
after
restriction
endont
tion.
We
applied
the
method
to
detect
nucleot
polymorphisms
in
human
genomic
DNA
and
c
the
mobility
shift
of
single-stranded
DNA
by
us
sequence
probe
arbitrarily
chosen.
Single-stran
the
same
nucleotide
length
can
be
separated
by
polyacryl-
amide
gel
electrophoresis,
probably
due
to
a
difference
in
their
predominant
semistable
conformations
(20).
The
mo-
bility
shift
of
single-stranded
DNAs
with
DNA
polymor-
phisms
observed
on
gel
electrophoresis
might
also
be
due
to
)~
g O
conformational
change,
and
so
we
designated
the
features
of
DNAs
as
SSCPs.
We
do
not
know
whether
nucleotide
8
9
substitution
at
any
position
in
a
fragment
can
be
detected
by
SSCP
analysis,
but
DNA
polymorphisms
at
a
variety
of
it
W
_
_
positions
in
a
fragment
could
cause
a
difference
in
its
conformation
and
result
in
change
in
mobility
of
the
single
df
vs/f
f/Vf
strands
on
gel
electrophoresis.
Therefore,
we
thought
that
DNA
polymorphism
could
be
detected
more
frequently
by
SSCP
analysis
than
by
RFLP
analysis,
and
our
experimental
_
_ _
results
revealed
that
this
was
in
fact
the
case.
Like
RFLP
analysis,
SSCP
analysis
is
simple
and
does
not
require
f
S/f
S/f
complicated
instruments
or
specialized
techniques.
As
we
confirmed
that
the
observed
SSCPs
were
due
to
allelic
variation
of
true
Mendelian
traits,
SSCP
analysis
of
4
*
*_
DNA
fragments
could
be
a
useful
and
simple
method
for
elucidating
the
human
genetic
linkage
map
by
studies
on
families.
Because
DNA
polymorphisms
have
been
estimated
s
s/s s/s
to
occur
once
every
few
hundred
nucleotides
of
the
human
genome
(1)
and
SSCP
analysis
can
reveal
nucleotide
substi-
tutions
at
various
positions
in
a
fragment,
any
restriction
endonuclease
fragment
with
a
nucleotide
length
suitable
for
strand
separation
may
provide
information
for
distinguishing
two
alleles.
Therefore,
in
theory,
on
a
nylon
membrane
carrying
separated
strands
of
all
possible
fragments
of
ge-
nomic
DNA,
DNA
polymorphisms
at
any
chromosomal
locus
can
be
detected
by
repeated
hybridization
of
the
members.
(A)
membrane
with
a
variety
of
probes.
indicated
at
the
SSCP
analysis
can
also
be
used
to
locate
genetic
elements
lalysis
using
the
involved
in
hereditary
diseases
and
to
detect
DNA
aberra-
ks
the
mobility
tions
in
human
cancers.
Comparison
of
DNA
fragments
from
(5
r.g)
digested
cancerous
portions
of
tissues
with
those
from
normal
por-
ing
32P-labeled
tions
by
SSCP
analysis
can
reveal
amplified
alleles
of
particular
genes
and
loss
of
heterozygosity
at
particular
chromosomal
loci.
A
remarkable
advantage
of
SSCP
analysis
;erved
at
17'C
is
that
it
can
be used
to
detect
point
mutations
at
various
tered
at
23TC.
positions
in
a
fragment.
Recently,
by
means
of
the
DNA
ne
semistable
polymerase
chain
reaction
(PCR),
a
DNA
segment
of
a
single
ng
buffer
also
cell
or
a
single
sperm
has
been
amplified
to
an
amount
;is
of
the
Pst
I
sufficient
for
analysis
by
hybridization
(36).
Our
preliminary
in
a
buffer
of
result
suggested
that
SSCP
analysis
of
DNA
segments
am-
H
8.3/2
mM
plified
by
PCR
technique
could
be
useful
for
diagnosis
of
iere
similar
to
genetic
aberrations.
;ence
of
1o
However
the
This
work
was
supported
in
part
by
a
grant-in-aid
from
the
Ministry
nobility
shifts
of
Health
and
Welfare
for
a
Comprehensive
10-Year
Strategy
for
inced
by
this
Cancer
Control,
Japan,
and
a
grant
from
the
Special
Coordination
rved
in
Fig.
2
Fund
of
the
Science
and
Technology
Agency
of
Japan.
M.O.
and
H.I.
formed
in
gel
were
recipients
of
Research
Resident
Fellowships
from
the
Foun-
mobility
shift
dation
for
Promotion
of
Cancer
Research.
l0o
glycerol
1.
Cooper,
D.
N.,
Smith,
B.
A.,
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H.
J.,
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&
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(1985)
Hum.
Genet.
69,
201-205.
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Botstein,
D.,
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L.,
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&
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Donis-Keller,
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in
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iclease
diges-
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&
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tide
sequence
(1987)
Cell
51,
319-337.
:ould
observe
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Gusella,
J.
F.,
Wexler,
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L.,
,ing
a
genomic
Anderson,
M.
A.,
Tanzi,
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E.,
Watkins,
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ided
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Wallace,
M.
R.,
Sakaguchi,
A.
Y.,
Young,
A.
B.,
Shoulson,
I.,
Genetics:
Orita
et
al.
.M-"

Proc.
Natl.
Acad.
Sci.
USA
86
(1989)
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E.
&
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J.
B.
(1983)
Nature
(London)
306,
234-
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Reeders,
S.
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